U.S. patent application number 10/050279 was filed with the patent office on 2002-11-14 for isolating cells expressing secreted proteins.
Invention is credited to Chen, Gang, Fandl, James P., Stahl, Neil, Yancopoulos, George D..
Application Number | 20020168702 10/050279 |
Document ID | / |
Family ID | 22995765 |
Filed Date | 2002-11-14 |
United States Patent
Application |
20020168702 |
Kind Code |
A1 |
Fandl, James P. ; et
al. |
November 14, 2002 |
Isolating cells expressing secreted proteins
Abstract
A method for identifying and isolating cells which produce
secreted proteins. This method is based upon a specific
characteristic or the expression level of the secreted protein by
transiently capturing the secreted protein on the surface of an
individual cell, allowing selection of rare cell clones from a
heterogeneous population. Also provided is the use of this method
to generate cells which produce a desired level of secreted protein
or secreted protein of a particular characteristic(s), and
organisms which possess such cells. In particular, the method
allows rapid isolation of high expression recombinant
antibody-producing cell lines, or may be applied directly to rapid
isolation of specific hybridomas, or to the isolation of
antibody-producing transgenic animals. This method is applicable
for any cell which secretes protein.
Inventors: |
Fandl, James P.;
(LaGrangeville, NY) ; Stahl, Neil; (Carmel,
NY) ; Chen, Gang; (Yorktown Heights, NY) ;
Yancopoulos, George D.; (Yorktown Heights, NY) |
Correspondence
Address: |
Laura J. Fischer
Regeneron Pharmaceuticals, Inc.
777 Old Saw Mill River Road
Tarrytown
NY
10591
US
|
Family ID: |
22995765 |
Appl. No.: |
10/050279 |
Filed: |
January 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60261999 |
Jan 16, 2001 |
|
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Current U.S.
Class: |
435/30 |
Current CPC
Class: |
C07K 2319/00 20130101;
C12N 15/1051 20130101; C07K 14/315 20130101; G01N 2500/10 20130101;
A01K 2217/05 20130101; C07K 16/2863 20130101; G01N 33/6845
20130101; C40B 30/04 20130101; C07K 14/52 20130101; A01K 2267/01
20130101; C07K 2317/77 20130101; G01N 33/56966 20130101 |
Class at
Publication: |
435/30 |
International
Class: |
C12Q 001/24 |
Claims
1. A method of detecting and isolating cells that produce any
secreted protein of interest (POI), comprising: a) constructing a
cell line transiently or stably expressing a cell surface capture
molecule, which binds the POI, by transfecting the cell line with a
nucleic acid that encodes such cell surface capture molecule; b)
transfecting said cell simultaneously or subsequently with a second
nucleic acid that encodes a POI wherein such POI is secreted; c)
detecting the surface-displayed POI by contacting the cells with a
detection molecule, which binds the POI; d) isolating cells based
on the detection molecule.
2. The method of claim 1 wherein the protein of interest is a
ligand, a soluble receptor protein, or a growth factor.
3. The method of claim 1 wherein the protein of interest is an
antibody, an Fab, a single chain antibody (ScFv), a fragment
thereof, or anything fused to an antibody constant region.
4. The method of claim 2 wherein the growth factor is selected from
the group consisting of Interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21,
Ciliary Neurotrophic Factor (CNTF), erythropoietin, Vascular
Endothelial Growth Factor (VEGF), angiopoietin 1, angiopoietin 2,
TNF, Interferon-gamma, GM-CSF, TGF.beta., TNF Receptor, fusion
proteins, and all approved therapies made in animal cells.
5. The method of claim 3 wherein the antibody is selected from the
group consisting of IgM, IgG, IgA, IgD or IgE, as well as various
subtypes of these.
6. The method of claim 1 wherein the nucleic acid that encodes a
POI is selected from a DNA library.
7. The method of claim 1 wherein the cell surface capture molecule
is a ligand-specific receptor, a receptor-specific ligand, an
antibody-binding protein, an antibody, an ScFv, a fragment thereof,
anything fused to a constant region of an antibody, and a peptide
from a phage display or peptide library and derivatives that bind
the POI.
8. The method of claim 7 wherein the cell surface capture molecule
is Ang1, And2, VEGF, Tie1, Tie2, VEGFRI (Flt1), VEGFRII (Flk1),
CNTF, CNTFR-alpha, cytokine receptor components, fusions of two or
more cytokine receptor components, or any fragments thereof.
9. The method of claim 7 wherein the antibody binding protein is an
Fc receptor, anti-immunoglobulin antibodies, anti-immunoglobulin
ScFv, Protein A, Protein L, Protein G, Protein H or functional
fragments thereof.
10. The method of claim 7 further comprising adding a membrane
anchor to a protein such that it remains anchored in a cell
membrane, exposed to the outside of the cell, and functions as a
cell surface capture molecule.
11. The method of claim 10 wherein the membrane anchor is a
transmembrane anchor or a GPI link.
12. The method of claim 10 wherein the membrane anchor may be
native to the cell, recombinant, or synthetic.
13. The method of claim 7 wherein a signal sequence is added to the
amino terminus of a protein, such that the protein is transported
to the cell surface, and functions as a cell surface capture
molecule.
14. The method of claim 13 wherein the signal sequence may be
native to the cell, recombinant, or synthetic.
15. The method of claim 1 wherein the isolated cell in claim 1(d)
is an antibody producing cell fused to an immortalized cell.
16. The method of claim 15 wherein the antibody producing cell is a
B-cell or derivative thereof.
17. The method of claim 16 wherein the B-cell derivative is a
plasma cell, a hybridoma, a myeloma, or a recombinant cell.
18. The method of claim 1 wherein the detection molecule(s) are two
molecules that bind each other and are differentially labeled.
19. The method of claim 1 wherein a blocking molecule which binds
the cell surface capture molecule or POI is added to reduce the
binding of the POI to a neighboring cell.
20. The method of claim 1 wherein the diffusion of the POI from the
expressing cell to a neighboring cell and its adherence to that
cell is reduced by increasing the viscosity of the media.
21. A non-human organism containing a cell produced by the method
of claim 1.
22. A non-human organism containing a cell produced by the method
of claim 1 wherein the cell surface capture molecule is specific
for antibodies.
23. A method of detecting and isolating cells that produce any
secreted protein of interest (POI), comprising: a) constructing a
cell line transiently or stably expressing a cell surface capture
molecule, which binds the POI, by transfecting the cell line with a
nucleic acid that encodes such cell surface capture molecule; b)
detecting a cell from (a) that expresses said cell surface capture
molecule; c) isolating and culturing the cell detected in (b); d)
transfecting said cell in (c) simultaneously or subsequently with a
second nucleic acid that encodes a POI wherein such POI is
secreted; e) detecting the surface-displayed POI by contacting the
cells with a detection molecule, which binds the POI; f) isolating
cells based on the detection molecule.
24. The method of claim 23 wherein the protein of interest is a
ligand, a soluble receptor protein, or a growth factor.
25. The method of claim 23 wherein the protein of interest is an
antibody, an ScFv, a fragment thereof, or anything fused to an
antibody constant region.
26. The method of claim 24 wherein the growth factor is selected
from the group consisting of Interleukin (IL)-1, IL-2, IL-4, IL-5,
IL-6, IL-7, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21,
Ciliary Neurotrophic Factor (CNTF), erythropoietin, Vascular
Endothelial Growth Factor (VEGF), angiopoietin 1, angiopoietin 2,
TNF, Interferon-gamma, GM-CSF, TGF.beta., TNF Receptor, fusion
proteins, and all approved therapies made in animal cells.
27. The method of claim 25 wherein the antibody is selected from
the group consisting of IgM, IgG, IgA, IgD or IgE, as well as
various subtypes of these.
28. The method of claim 23 wherein the nucleic acid that encodes a
POI is selected from a DNA library.
29. The method of claim 23 wherein the cell surface capture
molecule is a ligand-specific receptor, a receptor-specific ligand,
an antibody-binding protein, an antibody, an ScFv, a fragment
thereof, anything fused to a constant region of an antibody, and a
peptide from a phage display or peptide library and derivatives
that bind the POI.
30. The method of claim 29 wherein the cell surface capture
molecule is Tie1, Tie2, VEGFRI (Flt1), VEGFRII (Flk1), cytokine
receptor components or fusions of two or more cytokine receptor
components
31. The method of claim 29 wherein the antibody binding protein is
an Fc receptors, anti-immunoglobulin antibodies,
anti-immunoglobulin ScFv, Protein A, Protein L, Protein G, Protein
H or functional fragments thereof.
32. The method of claim 29 further comprising adding a membrane
anchor to a protein such that it remains anchored in a cell
membrane, exposed to the outside of the cell, and functions as a
cell surface capture molecule.
33. The method of claim 32 wherein the membrane anchor is a
transmembrane anchor or a GPI link.
34. The method of claim 32 wherein the membrane anchor may be
native to the cell, recombinant, or synthetic.
35. The method of claim 29 wherein a signal sequence is added to
the amino terminus of a protein, such that the protein is
transported to the cell surface, and functions as a cell surface
capture molecule.
36. The method of claim 35 wherein the signal sequence may be
native to the cell, recombinant, or synthetic.
37. The method of claim 23 wherein the isolated cell in (f) is an
antibody producing cell fused to an immortalized cell.
38. The method of claim 37 wherein the antibody producing cell is a
B-cell or derivative thereof.
39. The method of claim 38 wherein the B-cell derivative is a
plasma cell, a hybridoma, a myeloma, or a recombinant cell.
40. The method of claim 23 wherein the detection molecule(s) are
two molecules that bind each other and are differentially
labeled.
41. The method of claim 23 wherein a blocking molecule which binds
the cell surface capture molecule is added to reduce the diffusion
of the POI from the expressing cell to a neighboring cell.
42. The method of claim 23 wherein the diffusion of the POI from
the expressing cell to a neighboring cell and its adherence to that
cell is reduced by increasing the viscosity of the media.
43. A non-human organism containing a cell produced by the method
of claim 23.
44. A non-human organism containing a cell produced by the method
of claim 23 wherein the cell surface capture molecule is specific
for antibodies.
45. A method of detecting and isolating cells that produce a POI,
comprising: a) detecting a cell that expresses said cell surface
capture molecule in high yield; b) isolating and culturing the cell
detected in (a); c) transfecting said cell in (b) with a nucleic
acid that encodes a POI wherein such POI is secreted; d) detecting
the surface-displayed POI by contacting the cells with a detection
molecule which binds the POI; e) isolating cells based on the
detection molecule.
46. The method of claim 45 wherein the protein of interest is a
ligand, a soluble receptor protein, a growth factor, or an
antibody.
47. The method of claim 46 wherein the growth factor is selected
from the group consisting of Interleukin (IL)-1, IL-2, IL-4, IL-5,
IL-6, IL-7, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21,
Ciliary Neurotrophic Factor (CNTF), erythropoietin, Vascular
Endothelial Growth Factor (VEGF), angiopoietin 1, angiopoietin 2,
TNF, Interferon-gamma, GM-CSF, and TGF.beta..
48. The method of claim 46 wherein the antibody is selected from
the group consisting of IgM, IgG, IgA, IgD or IgE, as well as
various subtypes of these.
49. The method of claim 45 wherein the nucleic acid that encodes a
POI is selected from a DNA library.
50. The method of claim 45 wherein the cell surface capture
molecule is a ligand-specific receptor, a receptor-specific ligand,
or an antibody binding protein.
51. The method of claim 50 wherein the cell surface capture
molecule is Tie1, Tie2, VEGFRI (Flt1), VEGFRII (Flk1), cytokine
receptor components or fusions of two or more cytokine receptor
components.
52. The method of claim 50 wherein the antibody binding protein is
an Fc receptors, anti-immunoglobulin antibodies,
anti-immunoglobulin ScFv, Protein A, Protein L, Protein G, Protein
H, or functional fragments thereof.
53. The method of claim 45 further comprising adding a membrane
anchor to a protein such that it remains anchored in a cell
membrane, exposed to the outside of the cell, and functions as a
cell surface capture molecule.
54. The method of claim 53 wherein the membrane anchor is a
transmembrane anchor or a GPI link.
55. The method of claim 53 wherein the membrane anchor may be
native to the cell, recombinant, or synthetic.
56. The method of claim 50 wherein a signal sequence is added to
the amino terminus of a protein, such that the protein is
transported to the cell surface, and functions as a cell surface
capture molecule.
57. The method of claim 56 wherein the signal sequence may be
native to the cell, recombinant, or synthetic.
58. The method of claim 45 wherein the isolated cell in (f) is an
antibody producing cell fused to an immortalized cell.
59. The method of claim 58 wherein the antibody producing cell is a
B-cell or derivative thereof.
60. The method of claim 59 wherein the B-cell derivative is a
plasma cell, a hybridoma, a myeloma, or a recombinant cell.
61. The method of claim 45 wherein the detection molecule(s) are
two molecules that bind each other and are differentially
labeled.
62. The method of claim 45 wherein a blocking molecule which binds
the cell surface capture molecule is added to reduce the diffusion
of the POI from the expressing cell to a neighboring cell.
63. The method of claim 45 wherein the diffusion of the POI from
the expressing cell to a neighboring cell and its adherence to that
cell is reduced by increasing the viscosity of the media.
64. A non-human organism containing a cell produced by the method
of claim 45.
65. A non-human organism containing a cell produced by the method
of claim 45 wherein the cell surface capture molecule is specific
for antibodies.
66. A method of detecting and isolating cells that produce high
levels of POI, comprising: a) constructing a cell line expressing a
cell surface capture molecule, which binds the POI, by transfecting
the cell line with a nucleic acid that encodes such cell surface
capture molecule; b) detecting a cell from (a) that expresses said
cell surface capture molecule in high yield; c) isolating and
culturing those cells detected in (b) and allowing sufficient time
for said cell to secrete the POI; d) detecting the
surface-displayed POI by contacting the cells with (a) detection
molecule(s), one or more of which binds the POI; e) isolating cells
based on the detection molecule(s).
67. The method of claim 66 wherein the protein of interest is a
ligand, a soluble receptor protein, a growth factor, or an
antibody.
68. The method of claim 67 wherein the growth factor is selected
from the group consisting of Interleukin (IL)-1, IL-2, IL-4, IL-5,
IL-6, IL-7, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21,
Ciliary Neurotrophic Factor (CNTF), erythropoietin, Vascular
Endothelial Growth Factor (VEGF), angiopoietin 1, angiopoietin 2,
TNF, Interferon-gamma, GM-CSF, and TGF.beta..
69. The method of claim 67 wherein the antibody is selected from
the group consisting of IgM, IgG, IgA, IgD or IgE, as well as
various subtypes of these.
70. The method of claim 66 wherein the nucleic acid that encodes a
POI is selected from a DNA library.
71. The method of claim 66 wherein the cell surface capture
molecule is a ligand-specific receptor, a receptor-specific ligand,
or an antibody binding protein.
72. The method of claim 71 wherein the cell surface capture
molecule is Tie1, Tie2, VEGFRI (Flt1), VEGFRII (Flk1), cytokine
receptor components or fusions of two or more cytokine receptor
components
73. The method of claim 71 wherein the antibody binding protein is
an Fc receptors, anti-immunoglobulin antibodies,
anti-immunoglobulin ScFv, Protein A, Protein L, Protein G, Protein
H, or functional fragments thereof.
74. The method of claim 71 further comprising adding a membrane
anchor to a protein such that it remains anchored in a cell
membrane, exposed to the outside of the cell, and functions as a
cell surface capture molecule.
75. The method of claim 74 wherein the membrane anchor is a
transmembrane anchor or a GPI link.
76. The method of claim 74 wherein the membrane anchor may be
native to the cell, recombinant, or synthetic.
77. The method of claim 71 wherein a signal sequence is added to
the amino terminus of a protein, such that the protein is
transported to the cell surface, and functions as a cell surface
capture molecule.
78. The method of claim 77 wherein the signal sequence may be
native to the cell, recombinant, or synthetic.
79. The method of claim 66 wherein the isolated cell in (f) is an
antibody producing cell fused to an immortalized cell.
80. The method of claim 79 wherein the antibody producing cell is a
B-cell or derivative thereof.
81. The method of claim 66 wherein the B-cell derivative is a
plasma cell, a hybridoma, a myeloma, or a recombinant cell.
82. The method of claim 66 wherein the detection molecule(s) are
two molecules that bind each other and are differentially
labeled.
83. The method of claim 66 wherein a blocking molecule which binds
the cell surface capture molecule is added to reduce the diffusion
of the POI from the expressing cell to a neighboring cell.
84. The method of claim 66 wherein the diffusion of the POI from
the expressing cell to a neighboring cell and its adherence to that
cell is reduced by increasing the viscosity of the media.
85. A non-human organism containing a cell produced by the method
of claim 66.
86. A non-human organism containing a cell produced by the method
of claim 66 wherein the cell surface capture molecule is specific
for antibodies.
Description
[0001] This application claims priority to U.S. provisional
application No. 60/261,999, filed Jan. 16, 2001. Throughout this
application various publications are referenced. The disclosures of
these publications in their entireties are hereby incorporated by
reference into this application.
FIELD OF THE INVENTION
[0002] The field of this invention is a method for identifying and
isolating cells which produce secreted proteins. This method is
based upon a specific characteristic or the expression level of the
secreted protein by transiently capturing the secreted protein on
the surface of an individual cell, allowing selection of rare cell
clones from a heterogeneous population. The field also encompasses
the use of this method to generate cells which produce a desired
level of secreted protein or secreted protein of a particular
characteristic(s), and organisms which possess such cells. In
particular, the method allows rapid isolation of high expression
recombinant antibody-producing cell lines, or may be applied
directly to rapid isolation of specific hybridomas, or to the
isolation of antibody-producing transgenic animals. This method is
applicable for any cell which secretes protein.
Introduction
[0003] The method of the invention provides substantial advantages
over current methods for isolation and identification of
protein-secreting cells. The method described is more efficient,
more accurate, and more broadly applicable. Specifically, any cell
which secretes a protein may be isolated by the method of the
invention. This aspect is particularly important as many
therapeutic proteins are secreted. In addition, secreted
protein-producing cells may be isolated on the basis of the
protein's characteristics. For example, cells that secrete
antibodies may be rapidly and conveniently isolated based on
desired specificity, avidity, or isotype. Furthermore, the amount
of secreted protein produced may be directly quantified, unlike
many methods in the prior art wherein production of secreted
protein is indirectly quantified.
[0004] The present invention thus provides for a rapid, convenient,
and accurate method for systematically isolating protein-secreting
cells.
BACKGROUND OF THE INVENTION
[0005] Many proteins that are of potential pharmaceutical value are
secreted proteins, including growth factors, soluble receptor
domains, and most importantly monoclonal antibodies. Production
methods employing recombinant DNA technology to produce these and
other proteins use genetic expression systems which employ host
cells and expression vectors.
[0006] The expression vectors carry the gene of interest (GOI),
which is to be introduced, into the cell. These expression vectors
introduce genetic information, including the GOI(s), which
integrate into the host cell's own genetic material. Following
stable integration of the gene of interest (GOI), standard methods
for isolating high expression cells may involve collection of cell
pools, hand-picking colonies from plates, isolation of single cells
by limited dilution, or other methods known in the art. Pools or
individual clones are then expanded and screened for production of
the protein of interest (POI) by direct measurement of POI
activity, by immunological detection of POI, or by other suitable
techniques. These procedures are laborious, inefficient, expensive,
and the number of clones that can be analyzed is usually limited to
a few hundred.
[0007] The large degree of heterogeneity in protein expression by
cells following stable integration requires that many individual
clones be screened in an effort to identify the rare integration
event that results in a stable, high expression production cell
line. This requirement calls for methods that enable rapid
identification and isolation of cells expressing the highest level
of protein production. Moreover, the collection of clone pools, or
hand-picked colonies, risks losing high expression cells, which
often grow more slowly, to faster growing low expression cells.
Therefore, a need exists for methods that allow rapid screening and
isolation of individual cells capable of high level expression of a
secreted POI.
[0008] Incorporation of flow cytometry into methods used for the
isolation of stable expression cell lines has improved the
capability of screening large numbers of individual clones,
however, currently available methods remain inadequate for diverse
reasons. Early application of flow cytometry to the identification
and isolation of hybridomas with a defined specificity (Parks et
al. (1979) PNAS 76:1962, and Pallavacini et al. (1989) J. Immunol.
Methods 117:99), isotype (Dangl and Herzenberg (1982) J. Immunol.
Methods 52:1), or avidity (Jantscheff et al. (1988) J. Immunol.
141:1624) all depended on the detection of antibodies that were
non-specifically bound to the cell surface. These methods assumed a
correlation between the amount of surface bound and secreted
antibody. Diffusion of the POI between cells of different
characteristics was also a problem. Recently, two additional
methods that utilize flow cytometry have been developed for the
high throughput isolation of stable high expression cell lines.
[0009] The first method involves modification of the expression
plasmid to include a transcriptional read out for the GOI mRNA.
This is most often accomplished by inserting an internal ribosomal
entry site (IRES) and a gene whose protein product is easily
monitored by flow cytometry, most frequently green fluorescent
protein (GFP), between the stop codon of the GOI and the terminal
poly A site (Meng et al. (2000) Gene 242:201). The presence of an
IRES allows the POI and GFP to be translated from the same mRNA.
Therefore, the expression level of the GFP gene is indirectly
related to the mRNA level for the GOI. Clones that accumulate the
GFP at high levels are isolated by flow cytometry and then screened
for POI production. Because this method depends on the coupling of
GOI expression to the reporter gene by use of an IRES in a
recombinant construction, it is not applicable to the isolation of
hybridomas.
[0010] The use of flow cytometry in the isolation of expression
clones allows for the rapid analysis of large numbers of clones in
a high throughput format. Moreover, use of flow cytometry
significantly reduces the direct handling of cells. Unfortunately,
the level of GFP production is not a direct measure of the
production level of the POI. Various mechanisms may uncouple the
production of secreted POI from accumulation of GFP. Differences in
production of the POI and the GFP reporter may result from
differences in the translation efficiency of the two genes,
secretion efficiency of the POI, or stability of the polycistronic
mRNA.
[0011] Another method that uses flow cytometry to isolate
expression clones involves encapsulation of cells within agarose
microdrops (Weaver et al. (1990) Methods Enzymol. 2:234). In this
method biotinylated antibodies specific for the POI are bound to
the biotinylated agarose through streptavidin such that secreted
POI is captured and retained within the microdrop (Gray et al.,
(1995) J. Immunol. Methods 182:155). The trapped POI is detected by
immuno-staining with an antibody specific for the POI. To reduce
the encapsulating agarose from absorbing POI secreted from adjacent
cells, the cells are placed in a low-permeability medium. Those
cells with the highest antibody staining of the POI in the
embedding agarose are identified and isolated by flow cytometry.
The gel microdrop approach screens cells directly for their ability
to secrete POI, rather than indirectly screening for expression of
GOI mRNA, but requires the availability of suitable antibodies for
trapping and staining the secreted POI and the procedure requires
special equipment to generate the agarose gel microdrops. Moreover,
some cells may be sensitive to the encapsulation process.
[0012] A variation of this method circumvents the requirement for
embedding cells in a matrix by directly binding an antibody,
specific for the POI, to the cell surface (Manz et al. 1995. PNAS
92:19211925). In this method, non-specific biotinylation of cell
surface proteins with biotin-hydroxysuccinimide ester is followed
by contact with a streptavidin-conjugated antibody capable of
binding the POI. Cells secreting the POI become decorated with the
POI which is then detected with an appropriately labeled second
antibody. However, diffusion of POI between neighboring cells is
problematic, and this method also requires a high viscosity medium
to reduce diffusion of POI away from expressing cells. Because
these high viscosity media are required for discriminating cells,
the cells must be washed and placed in a medium suitable for cell
sorting if so desired.
[0013] The problems associated with identification and isolation of
high expression recombinant cell lines especially applies to the
isolation of hybridomas that express an antibody of interest.
However, the identification of useful hybridomas includes several
additional problems; they must be screened first for
antigen-binding activity, then for immunoglobulin isotype.
Moreover, GFP-based methods are not applicable to the
identification and isolation of hybridomas because construction of
hybridomas does not include a recombinant construct such that
expression of the antibody genes can be linked to a transcriptional
reporter such as GFP. Hybridoma screening is a slow, laborious
endeavor where the number of clones screened is limited by existing
technologies.
[0014] A similar problem involves the selection of rare cells
producing an antibody, an ScFv, a fragment thereof, or anything
fused to an antibody constant region, with a desired specificity,
isotype, and avidity for a particular antigen, from a heterogeneous
population of cells expressing different antibodies, ScFvs,
fragments thereof, or anything fused to antibody constant
regions.
[0015] Thus a need exists for a rapid and efficient method of
identifying and isolating cells expressing various secreted POIs
from a large population of cells. Most desirable is a method which
measures the protein expression level rather than the mRNA, as the
measure of mRNA often does not accurately reflect the levels of
protein which will ultimately be produced. In addition, a need
exists for a more efficient method to identify cells that produce
particular antibodies than what is currently available in the
art.
SUMMARY OF THE INVENTION
[0016] The present invention describes a high-throughput screening
method for the rapid isolation of those cells that secrete protein
by directly screening for the POI. This invention also allows for
the convenient monitoring of POI expression on a single-cell basis
during the manufacturing process. Furthermore, this technology can
be directly applied to screening of antibody producing cells.
[0017] This invention relates to the construction of cell lines
that express cell surface capture molecules that bind various
secreted POIs, and the use thereof to identify and isolate the
cells that secrete the POI. Isolation of a cell by the methods of
this invention may be based on the expression level of the POI or a
specific characteristic of the POI. Through the construction or use
of such a cell, any secreted protein may be captured by the cell
surface capture molecule provided there is a corresponding affinity
between the two.
[0018] As explained further, any molecule may be manipulated such
that it can be used as a cell surface capture molecule. Therefore,
this invention may be utilized to isolate any cell which secretes a
protein. Furthermore, many cells may be transfected to produce
secreted proteins, and therefore even cells that do not secrete
proteins in their native state may be isolated as secreted protein
producers through the application of this invention.
[0019] Detection of the cells with the displayed POI may be
accomplished through the use of any molecule capable of directly or
indirectly binding the displayed POI. Such detection molecules may
facilitate the detection and/or isolation of the cells displaying
the POI. In one embodiment, two molecules that bind each other and
are deferentially labeled are utilized. The detection and/or
isolation may be accomplished through standard techniques known in
the art.
[0020] Additionally, this invention may be applied to the isolation
of antibody-producing cells. Specifically, an antibody producing
cell may be fused to a immortalized cell which expresses a cell
surface capture molecule which binds the POI, which is an antibody
in this case. The antibody-producing cell may be a B-cell or a
derivative thereof, such as a plasma cell, a hybridoma, a myeloma,
or a recombinant cell. The invention can also be used for isolation
of cells that express desired levels, specifically high levels, of
a recombinant antibody or fragments thereof. This invention also
allows for the isolation of rare cells expressing an antibody,
ScFv, fragments thereof, or anything fused to an antibody constant
region, with a desired specificity, isotype, and avidity for a
particular antigen from a population of heterologous cells
expressing a library of antibody genes with varying binding
specificity, isotype, and avidity. More specifically, the invention
relates to the identification of antibody-producing cells that
express secreted antibodies of a desired specificity and isotype,
as well as antibodies that are specific for a desired epitope.
[0021] In another embodiment of the invention, transgenic animals
may be created that express a cell surface capture molecule. The
cells from such transgenic animal may then be screened directly for
the production of the POI.
[0022] The invention also relates to a method of detecting and
isolating cells that produce any secreted protein of interest
(POI), comprising:
[0023] a) constructing a cell line transiently or stably expressing
a cell surface capture molecule, which binds the POI, by
transfecting the cell line with a nucleic acid that encodes such
cell surface capture molecule;
[0024] b) transfecting said cell simultaneously or subsequently
with a second nucleic acid that encodes a POI wherein such POI is
secreted;
[0025] c) detecting the surface-displayed POI by contacting the
cells with a detection molecule, which binds the POI;
[0026] d) isolating cells based on the detection molecule.
[0027] The invention further relates to a method of detecting and
isolating cells that produce any secreted POI, comprising:
[0028] a) constructing a cell line transiently or stably expressing
a cell surface capture molecule which binds the POI by transfecting
with a nucleic acid that encodes such cell surface capture
molecule;
[0029] b) detecting a cell from (a) that expresses said cell
surface capture molecule;
[0030] c) isolating and culturing those cells detected in (b);
[0031] d) transfecting said cell in (c) simlutaneoulsy or
subsequently with a second nucleic acid that encodes a POI wherein
such protein is secreted;
[0032] e) detecting the surface-displayed POI by contacting the
cells with (a) detection molecule(s), one or more of which binds
the POI;
[0033] f) isolating cells based on the detection molecule(s).
[0034] The invention still further relates to a method of detecting
and isolating cells that produce any secreted POI, comprising:
[0035] a) detecting a cell that transiently or stably expresses
said cell surface capture molecule in high yield;
[0036] b) isolating and culturing those cells detected in (a);
[0037] c) transfecting said cell in (b) with a nucleic acid that
encodes a POI wherein such protein is secreted;
[0038] d) detecting the surface-displayed POI by contacting the
cells with (a) detection molecule(s), one or more of which binds
the POI;
[0039] e) isolating cells based on the detection molecule(s).
[0040] The invention further relates to a method of detecting and
isolating cells that produce any secreted POI, comprising:
[0041] a) constructing a cell line expressing a cell surface
capture molecule which binds the POI by transfecting with a nucleic
acid that encodes such cell surface capture molecule;
[0042] b) detecting a cell from (a) that expresses said cell
surface capture molecule in high yield;
[0043] c) isolating and culturing those cells detected in (b) and
allowing sufficient time for said cell to secrete the POI;
[0044] d) detecting the surface-displayed POI by contacting the
cells with (a) detection molecule(s), one or more of which binds
the POI;
[0045] e) isolating cells based on the detection molecule(s).
[0046] The invention relates to a non-human organism containing a
cell produced by these methods. Specifically, such non-human
organism may contain a cell with a cell surface capture molecule
specific for antibodies.
[0047] In addition, the invention contemplates adding a membrane
anchor to a protein such that it remains anchored in a cell
membrane, exposed to the outside of the cell, and functions as a
cell surface capture molecule. Such membrane anchor may be a
transmembrane anchor or a GPI link, and may be native to the cell,
recombinant, or synthetic.
[0048] The invention also embodies the addition of a signal
sequence to the amino terminus of a protein, such that the protein
is transported to the cell surface, and functions as a cell surface
capture molecule. Such signal sequence may be native to the cell,
recombinant, or synthetic.
[0049] In another embodiment, a blocking molecule which binds the
cell surface capture molecule is added to reduce the diffusion of
the POI from the expressing cell to a neighboring cell. In an
additional embodiment, the diffusion of the POI from the expressing
cell to a neighboring cell and its adherence to that cell is
reduced by increasing the viscosity of the media.
[0050] This invention further relates to the identification and
selection of cells that express secreted proteins including
ligands, soluble receptor proteins, growth factors, and antibodies.
Such secreted proteins may be recombinantly produced or naturally
occurring. In addition, the nucleic acid that encodes a POI may be
selected from a library, including but not limited to a cDNA
library or a genomic library.
[0051] In one embodiment, such growth factors may be selected from
the group consisting of Interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21,
Ciliary Neurotrophic Factor (CNTF), erythropoietin, Vascular
Endothelial Growth Factor (VEGF), angiopoietin 1, angiopoietin 2,
TNF, Interferon-gamma, GM-CSF, TGF.beta., TNF Receptor, fusion
proteins, and all approved therapies made in animal cells.
[0052] In another embodiment, the antibody is selected from the
group consisting of IgM, IgG, IgA, IgD or IgE, as well as various
subtypes of these.
[0053] In yet another embodiment, the invention employs a
ligand-specific receptor, a receptor-specific ligand, or an
antibody binding protein, as the cell surface capture molecule
which binds the POI. Such cell surface capture molecule may be
recombinantly produced or naturally occurring.
[0054] In one embodiment, the cell surface capture molecule is a
ligand-specific receptor, a receptor-specific ligand, an
antibody-binding protein, an antibody, an ScFv, a fragment thereof,
anything fused to a constant region of an antibody, and a peptide
from a phage display or peptide library and derivatives that bind
the POI. In another embodiment, the cell surface capture molecule
is selected from the group consisting of Tie1, Tie2, VEGFRI (Flt1),
VEGFRII (Flk1), cytokine receptor components or fusions of two or
more cytokine receptor components.
[0055] In addition the invention relates to the identification of
B-cells and derivatives thereof, or hybridomas that express
secreted antibodies of a desired specificity, affinity or isotype.
The invention can also be used for isolation of cells that express
desired levels of an antibody or antibody fragments.
[0056] The invention further relates to the use of
anti-immunoglobulin antibodies, anti-immunoglobulin ScFv, Protein
A, Protein L, Protein G, or Fc receptor (FcR) as the cell surface
capture molecule that binds the POI, wherein the POI is a secreted
antibody.
BRIEF DESCRIPTION OF THE FIGURES
[0057] FIG. 1 represents the construction of pTE084, designed for
the constitutive expression of human Fc.gamma.RI from the upstream
CMV-MIE promoter.
[0058] FIG. 2A shows a flow cytometry single parameter histogram of
unstained CHO K1 cells.
[0059] FIG. 2B shows a flow cytometry single parameter histogram of
FITC-hFc stained CHO K1.
[0060] FIG. 2C shows a flow cytometry single parameter histogram of
FITC-hFc stained G418-resistant CHO K1 cell pool after pTE084
transfection.
[0061] FIG. 2D shows a flow cytometry single parameter histogram of
FITC-hFc stained RGC3 cells.
[0062] FIG. 3 summarizes the ability of IgG from a variety of
animal species to block 4SC622 from binding to RGC1 cells.
[0063] FIG. 4A shows a flow cytometry single parameter histogram of
unstained RGC1 cells.
[0064] FIG. 4B shows a flow cytometry single parameter histogram of
4SC622 binding to Fc.gamma.RI-expressing RGC1 cells as indicated by
PE-AG184 binding.
[0065] FIG. 4C shows a flow cytometry single parameter histogram of
rat IgG blocking the binding of 4SC622 to RGC1 cells as indicated
by loss of PE-AG184 binding.
[0066] FIG. 5A shows a flow cytometry dual parameter histogram of
RGC2 cells, which express the gene for hFc.gamma.RI and GFP,
stained with PE-AG184.
[0067] FIG. 5B shows a flow cytometry dual parameter histogram of
RGC2 cells, which express the gene for hFc.gamma.RI and GFP,
incubated with 1 ug/ml 4SC622 for 18 hours before being stained
with PE-AG184.
[0068] FIG. 5C shows a flow cytometry dual parameter histogram of
RGC4 cells, which express the gene for hFc.gamma.RI and 4SC622,
stained with PE-AG184.
[0069] FIG. 5D shows a flow cytometry dual parameter histogram of
RGC4 cells, which express the gene for hFc.gamma.RI and 4SC622,
incubated with rat IgG (1 mg/ml) for 18 hours before being stained
with PE-AG184.
[0070] FIG. 5E shows a flow cytometry dual parameter histogram of a
mixture of RGC2, which express the gene for hFc.gamma.RI and GFP,
and RGC4 cells, which express the gene for hFc.gamma.RI and 4SC622,
mixed and incubated together for 18 hours prior to staining with
PE-AG184.
[0071] FIG. 5F shows a flow cytometry dual parameter histogram of a
mixture of RGC2, which express the gene for hFc.gamma.RI and GFP,
and RGC4 cells, which express the gene for hFc.gamma.RI and 4SC622,
mixed and incubated together for 18 hours with 1 mg/ml rat IgG
prior to staining with PE-AG184.
[0072] FIG. 6 shows a flow cytometry single parameter histogram of
a MSX-resistant pool of RGC1 cells transfected with pEE14.1-622.
Cells in the top 3% gate (R3), 7-11% gate (R5), and 15-19% gate
(R7) were collected, expanded, and their 4SC622 productivity
quantitated by immuno-staining.
[0073] FIG. 7 summarizes a comparison of the specific
productivities of 4SC622 expressing cell lines. CHO K1 transiently
transfected with pEE14.1-622, hand-picked stable MSX-resistant
clones of CHO K1 transfected with pEE14.1-622, and MSX-resistant
4SC622 production clones isolated after transfection of RGC1 cells
with pEE14.1-622.
[0074] FIG. 8A shows a flow cytometry single parameter histogram of
RGC1 cells stained with PE-AG184.
[0075] FIG. 8B shows a flow cytometry single parameter histogram of
RGC1 cells incubated with 1 ug/ml 4SC622 for 1 hour prior to being
stained with PE-AG184.
[0076] FIG. 8C shows a flow cytometry single parameter histogram of
RGC1 cells that were incubated with 1 ug/ml 4SC622 for 1 hour, then
incubated in medium without 4SC622 for 18 hours prior to staining
with PE-AG184.
[0077] FIG. 9 shows that expression of the gene for hFc.gamma.RI
results in loss of 4SC622 from the culture medium. RGC1 cells, or
CHO K1 parental cells, were incubated in medium containing 2 ug/ml
4SC622. The concentration of 4SC622 remaining in the media was
quantitated by immuno-staining after 24 hours, and 72 hours
incubation.
[0078] FIG. 10 represents the construction of pTE158, designed to
allow TetR-regulated expression of human Fc.gamma.RI. Two repeats
of the tet operator sequence (TetO) are immediately downstream of
the CMV promoter in pTE158.
[0079] FIG. 11A shows a flow cytometry single parameter histogram
of CHO K1 cells stained with FITC-hFc.
[0080] FIG. 11B shows a flow cytometry single parameter histogram
of RGC10 cells stained with FITC-hFc.
[0081] FIG. 11C shows a flow cytometry single parameter histogram
of RGC10 cells induced with 1 ug/ml doxycycline for three days
prior to staining with FITC-hFc.
[0082] FIG. 12A shows a flow cytometry single parameter histogram
of CHO K1 cells stained with polyclonal FITC-conjugated anti-human
IgG (H+L) F(ab').sub.2 fragment.
[0083] FIG. 12B shows a flow cytometry single parameter histogram
of MSX-resistant RGC10 cells transfected with pEE14.1-622 and
incubated with rat IgG (1 mg/ml) for 18 hours prior to staining
with polyclonal FITC-conjugated anti-human IgG (H+L) F(ab').sub.2
fragment.
[0084] FIG. 12C shows a flow cytometry single parameter histogram
of MSX-resistant RGC10 cells transfected with pEE14.1-622 induced
with 1 ug/ml doxycycline for three days then incubated with rat IgG
(1 mg/ml) for 18 hours prior to staining with polyclonal
FITC-conjugated anti-human IgG (H+L) F(ab').sub.2 fragment.
[0085] FIG. 13 summarizes the specific productivities of
MSX-resistant stable clones of RGC10 cells transfected with
pEE14.1-622.
[0086] FIG. 14 represents the structure of pTE255, designed for the
constitutive expression of human Fc.gamma.RI from the upstream
MoMuSV LTR promoter.
[0087] FIG. 15A shows a flow cytometry single parameter histogram
of unstained Sp2/0 cells.
[0088] FIG. 15B shows a flow cytometry single parameter histogram
of Cy5-hFc stained Sp2/0 cells.
[0089] FIG. 15C shows a flow cytometry single parameter histogram
of unstained Sp2/0-FcR-4 cells.
[0090] FIG. 15D shows a flow cytometry single parameter histogram
of Cy5-hFc stained Sp2/0-FcR-4 cells.
[0091] FIG. 16 represents the structure of pTE209, designed for the
constitutive expression of 4SC622 from the upstream CMV MIE
promoter.
[0092] FIG. 17A shows a flow cytometry single parameter histogram
of unstained hygromycin B-resistant Sp2/0-FcR-4 cells transfected
with pTE209.
[0093] FIG. 17B shows a flow cytometry single parameter histogram
of hygromycin B-resistant Sp2/0-FcR-4 cells transfected with pTE209
and incubated with rabbit IgG (1 mg/ml) for 18 hours prior to
staining with polyclonal FITC-conjugated anti-human IgG (H+L)
F(ab').sub.2 fragment.
[0094] FIG. 17C shows a flow cytometry single parameter histogram
of unstained cells expanded from the top 1% most fluorescent cells
in FIG. 4B.
[0095] FIG. 17D shows a flow cytometry single parameter histogram
of the cells expanded from the top 1% most fluorescent cells in
FIG. 4B, incubated with rabbit IgG (1 mg/ml) for 18 hours prior to
staining with polyclonal FITC-conjugated anti-human IgG (H+L)
F(ab').sub.2 fragment.
[0096] FIG. 17E shows a flow cytometry single parameter histogram
of unstained clone 5H11 cells.
[0097] FIG. 17F shows a flow cytometry single parameter histogram
of clone 5H11 cells incubated with rabbit IgG (1 mg/ml) for 18
hours prior to staining with polyclonal FITC-conjugated anti-human
IgG (H+L) F(ab').sub.2 fragment.
[0098] FIG. 18 shows schematic diagrams of domains of Protein G and
Protein G/hFc.gamma.RI fusion protein encoded in pTE300.
[0099] FIG. 19 is an outline of the construction of pTE300,
designed for the expression of a chimeric protein containing the
RORI signal sequence, the Fc binding domain of Protein G, and the
transmembrane and intracellular domain of hFc.gamma.RI from the
upstream CMV MIE promoter.
[0100] FIG. 20A shows a flow cytometry single parameter histogram
of unstained RGC14 cells.
[0101] FIG. 20B shows a flow cytometry single parameter histogram
of FITC-hFc stained RGC14 cells.
[0102] FIG. 20C shows a flow cytometry single parameter histogram
of unstained G418-resistant RGC14 cell pool transfected with
pTE300.
[0103] FIG. 20D shows a flow cytometry single parameter histogram
of FITC-hFc stained G418-resistant RGC14 cell pool transfected with
pTE300.
[0104] FIG. 20E shows a flow cytometry single parameter histogram
of unstained RGC18 cells.
[0105] FIG. 20F shows a flow cytometry single parameter histogram
of RGC18 cells incubated with 10% fetal bovine serum for 2 hours
prior to staining with polyclonal FITC-conjugated anti-bovine IgG
(H+L) F(ab').sub.2 fragment.
[0106] FIG. 21A shows a flow cytometry single parameter histogram
of unstained hygromycin B-resistant cell pool derived from RGC18
after transfection with pTE209.
[0107] FIG. 21B shows a flow cytometry single parameter histogram
of the hygromycin B-resistant cell pool derived from RGC18 after
transfection with pTE209, incubated with rabbit IgG (1 mg/ml) for
18 hours prior to staining with polyclonal FITC-conjugated
antihuman IgG (H+L) F(ab').sub.2 fragment.
[0108] FIG. 22A shows a flow cytometry single parameter histogram
of unstained RGC18 cells.
[0109] FIG. 22B shows a flow cytometry single parameter histogram
of RGC18 cells incubated with 4SC622 (1 .mu.g/ml) for 1 hour prior
to staining with polyclonal FITC-conjugated anti-human IgG (H+L)
F(ab').sub.2 fragment.
[0110] FIG. 22C shows a flow cytometry single parameter histogram
of RGC18 cells incubated with 4SC622 (1 .mu.g/ml) and rabbit IgG (1
mg/ml) for 1 hour prior to staining with polyclonal FITC-conjugated
anti-human IgG (H+L) F(ab').sub.2 fragment.
[0111] FIG. 22D shows a flow cytometry single parameter histogram
of RGC18 cells incubated with 4SC622 (1 .mu.g/ml) for 18 hours
prior to staining with polyclonal FITC-conjugated anti-human IgG
(H+L) F(ab').sub.2 fragment.
[0112] FIG. 22E shows a flow cytometry single parameter histogram
of RGC18 cells incubated with 4SC622 (1 .mu.g/ml) for 2 hours then
with 4SC622 (1 .mu.g/ml) and rabbit IgG (1 mg/ml) for 18 hours
prior to staining with polyclonal FITC-conjugated anti-human IgG
(H+L) F(ab').sub.2 fragment.
[0113] FIG. 22F shows a flow cytometry single parameter histogram
of unstained RGC19 cells derived from RGC18 cells by transfection
with pTE209.
[0114] FIG. 22G shows a flow cytometry single parameter histogram
of RGC19 cells stained with polyclonal FITC-conjugated anti-human
IgG (H+L) F(ab').sub.2 fragment.
[0115] FIG. 22H shows a flow cytometry single parameter histogram
of RGC19 cells incubated with rabbit IgG (1 mg/ml) for 18 hours
prior to staining with polyclonal FITC-conjugated anti-human IgG
(H+L) F(ab').sub.2 fragment.
DETAILED DESCRIPTION OF THE INVENTION
[0116] This invention describes a novel and previously unknown
method of identifying and isolating the cells which produce
secreted proteins. The invention is based on the production of a
cell line that expresses a molecule, localized to the cell surface,
which binds the POI. The cell surface-displayed POI can then be
detected by labeling with various detection molecules. The amount
of POI displayed on the cell surface, under specific conditions, is
a direct measure of the total amount of POI secreted. POI producers
may then be isolated from non-producers, and levels of production
or POI characteristics may be differentiated. The advantage of the
invention is that it directly quantifies the secreted POI rather
than indirectly measuring the mRNA.
[0117] This invention relates to the construction or use of cells
that express cell surface capture molecules which bind various
secreted POIs in the same cell that produces the POI. As the cell
secretes the POI, these cell surface capture molecules bind it, or
complexes of POI and cell surface capture molecules may form
intracellularly and then get secreted. Binding may occur in an
autocrine manner or while being secreted. The cells that produce
the secreted POI may then be identified and isolated. Such
identification and isolation may be based on characteristics of the
POI, production of the POI or lack thereof, or by specified levels
of production. The cell surface capture molecule and/or the POI may
be produced by the cell in its native state, or the cell surface
capture molecules and/or the POI may be recombinantly produced.
Through the construction or use of such a cell, any secreted
protein may be captured by the cell surface capture molecule
provided there is a corresponding affinity between the two. As
explained further, any molecule may be manipulated such that it can
be used as a cell surface capture molecule. Therefore, this
invention may be utilized to isolate any cell which secretes a
protein.
[0118] Most any protein has the capacity to function as a cell
surface capture molecule as described by the invention. What is
necessary is the ability of the desired protein to be anchored to
the cell membrane and exposed to the extracellular space. If the
desired cell has a signal sequence then only a membrane anchor,
including but not limited to a transmembrane anchor or a GPI
linkage signal, need be added to the cell surface capture molecule
such that it remains anchored in the cell membrane exposed to the
outside of the cell. Furthermore, if the desired protein lacks a
signal sequence, a signal sequence may be added to the amino
terminus of the desired protein, such that it is transported to the
cell surface. A signal sequence and a membrane anchor may be native
to the cell, recombinant, or synthetic.
[0119] Cells often secrete a wide variety of proteins, endogenously
or following the introduction of recombinant DNA. Any secreted
protein may be identified and the cell producing it may be isolated
according to the method of this invention. Such secreted proteins
include but are not limited to growth factors, growth factor
receptors, ligands, soluble receptor components, antibodies, and
peptide hormones. Such secreted proteins may or may not be
recombinant. That is, the secretion of some proteins of interest
from the desired cell may not require the introduction of
additional nucleotide sequences. For example, the secretion of
antibodies from B-cells or plasma cells is not the result of
introduction of recombinant nucleotide sequences into the B-cell or
plasma cell. Recombinant secreted proteins may be produced by
standard molecular biology techniques well known to the skilled
artisan (see e.g., Sambrook, J., E. F. Fritsch And T. Maniatis.
Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2,
and 3, 1989; Current Protocols in Molecular Biology, Eds. Ausubel
et al., Greene Publ. Assoc., Wiley Interscience, NY). These
secreted proteins are useful for many commercial and research
purposes. This invention encompasses the production of such
secreted proteins through the methodologies of the invention.
Detection of the cells with the displayed POI may be accomplished
through the use of any molecule capable of directly or indirectly
binding the displayed POI. Such detection molecules may facilitate
the detection and/or isolation of the cells displaying the POI.
[0120] The invention is applicable to the isolation of inter alia,
a) ligand-producing cells by using the ligand-specific receptor as
the cell surface capture molecule, b) soluble receptor-producing
cells by using a surface bound receptor-specific ligand as the cell
surface capture molecule, or c) antibody-producing cells by using
an antibody-binding protein as the cell surface capture
molecule.
[0121] In one embodiment, the invention is applicable to the
isolation of ligand-producing cells by using the ligand-specific
receptor as the cell surface capture molecule. More specifically,
cells can be used or constructed that express a cell surface
capture molecule capable of binding a growth factor or a cytokine.
These cell surface capture molecule may include, but are not
limited to Tie1, Tie2, VEGFRI (Flt1), VEGFRII (Flk1), cytokine
receptor components or fusions of two or more cytokine receptor
components. Such receptors may bind a POI, such as cytokine or a
growth factor. The cytokines and growth factors may include, but
are not limited to, Interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21,
Ciliary Neurotrophic Factor (CNTF), erythropoietin, Vascular
Endothelial Growth Factor (VEGF), angiopoietin 1, angiopoietin 2,
TNF, Interferon-gamma, GM-CSF, and TGF.beta..
[0122] In another embodiment, the invention is applicable to the
isolation of antibody-producing cells by using an antibody-binding
protein as the cell surface capture molecule. In one embodiment, an
antibody producing cell may be fused to an immortalized cell which
expresses a cell surface capture molecule which binds the POI which
is an antibody in this case. Such antibody producing cells include
but are not limited to B-cells and derivatives thereof, especially
plasma cells, hybridomas (e.g. NSO), myelomas (e.g. SP2/0),
recombinant cells (e.g. CHO cells expressing recombinant
antibodies), or other cells producing fusion proteins consisting of
antibody fragments, such as, for example, the Fc fragment from IgG.
The POI may be an antibody produced by such cells which include but
are not limited to IgM, IgG, IgA, IgD and IgE, as well as various
subtypes of these. Cell surface capture molecules that may be used
to bind antibodies include, but are not limited to, Fc receptors,
such as Fc gamma RI, Fc gamma RII, Fc gamma RIII,
anti-immunoglobulin antibodies, Protein A, Protein L, Protein G,
and Protein H, or functional fragments thereof.
[0123] The invention, as applied to the isolation of
antibody-producing cells, could be used to identify and isolate
cells that express antibodies with a desired specificity, isotype,
and avidity. Moreover, detection with a peptide or a protein
fragment would allow identification and isolation of antibody
producing cells that express antibodies specific for a desired
epitope.
[0124] In accordance with the methodology of this invention, a cell
is first transfected with a vector containing a nucleotide sequence
that encodes a cell surface capture molecule that is capable of
binding the secreted POI, under conditions in which such cell
surface capture molecule is expressed. Transfected cells which are
appropriate producers of such cell surface capture molecules are
then detected and isolated, and such cells are cultured. These
cells may either naturally produce the POI, or the POI may be
recombinantly produced. If the cells naturally produce the POI,
they are ready for detection and isolation. If the POI is to be
recombinantly produced, then the isolated and cultured cells
expressing the specified cell surface capture molecule are
transfected with second nucleotide sequence that encodes the
secreted POI, under conditions in which the secreted POI is
expressed. Upon expression, the secreted POI binds to the cell
surface capture molecules and the cells displaying bound POI are
detected and isolated.
[0125] If the POI is naturally produced by the cell, the cell will
not be transfected with nucleotide sequence encoding the POI.
Therefore, this aspect of the invention is applicable to any and
all cells producing a POI. In addition, if the cell surface capture
molecule is naturally produced by the cell, the cell need not be
transfected with nucleotide sequences encoding the cell surface
capture molecule. Therefore, this aspect of the invention is
applicable to any and all cells producing a cell surface capture
molecule.
[0126] Transfection and expression are effected through the use of
various vectors comprising different expression cassettes and
promoters, either constitutive or regulated, and may also include
enhancers, transcription terminators, splice donor and acceptor
sites and other nucleotide sequences (Kaufman et al., (1991) Meth.
Enzymology 185:487).
[0127] These vectors are generally commercially available or can be
readily prepared by standard techniques known in the art and
provide for expression in a host either by maintenance as an
extrachromosomal element or by integration into the host genome.
For a mammalian host, a wide variety of vectors are known based on
viral replication systems, such as Simian virus, bovine papilloma
virus, adenovirus, Epstein Barr Virus, retrovirus, and the like.
These vectors can be used as expression vectors where
transcriptional and translational initiation and termination
signals are present and one or more restriction sites are available
for insertion of a GOI. In addition, the vectors normally have one
or more markers which allow for selection of host cells which
contain the expression vector. The marker may provide for
prototrophy to an auxotrophic host; biocide resistance, e.g.,
resistance to antibiotics, such as G418, or heavy metals, such as
copper; or the like. If desired, expression vectors can be prepared
by joining the various components, such as the replication system,
markers, and transcriptional and translational regulatory
initiation and termination signals in conjunction with the GOI.
Frequently, a vector will include a prokaryotic replication system,
which allows for cloning, manipulation, purification, and expansion
of the desired DNA sequence.
[0128] A wide variety of transcriptional and translational
regulatory sequences may be employed, depending upon the nature of
the host. The transcriptional and translational regulatory signals
may be derived from viral sources, such as cytomegalovirus,
adenovirus, bovine papilloma virus, Simian virus, or the like,
where the regulatory signals are associated with a particular gene
which has a high level of expression. Alternatively, promoters from
mammalian expression products, such as actin, collagen, and myosin,
may be employed. Transcriptional initiation regulatory signals may
be selected which allow for repression or activation, so that
expression of the fused genes can be modulated. Regulatory signals
may be derived from natural sources or may be chimeric in that
sequences with the regulatory signal may originate from different
sources.
[0129] Furthermore, the nucleic acid sequences employed for
preparation of the vectors may be derived from a variety of
sources. These sources include genomic DNA, cDNA, synthetic DNA,
and combinations thereof. The genomic DNA may or may not include
naturally occurring introns. The DNA obtained from natural sources,
namely the genomic DNA or cDNA, may be obtained in a variety of
ways. Host cells coding for the desired sequence may be isolated,
the genomic DNA may be fragmented, conveniently by one or more
restriction endonucleases, and the resulting fragments may be
cloned and screened with a probe for the presence of the DNA
sequence coding for the polypeptide sequence of interest. Once the
cloned fragment has been identified which contains the desired DNA
sequence, this fragment may be further manipulated to remove
superfluous DNA, modify one or both termini, remove all or a
portion of intervening sequences (introns), or the like.
[0130] Once the vector has been prepared for expression, it may be
introduced into an appropriate host. Various techniques may be
employed to introduce nucleic acids into host cells including, but
not limited to, calcium phosphate coprecipitation,
DEAE-dextran-mediated transfection, electroporation,
polybrene-mediated transfection, lipid-mediated transfection,
lipofection, DNA microinjection, and microprojectile-mediated gene
transfer (Keown et al., (1990) Meth. Enzymology 185:527.)
[0131] The skilled artisan will be aware that well known techniques
exist in the art which induce stable integration of GOI and
subsequent expression of proteins to be identified and isolated for
further use. Kaufman et al., ((1991) Meth. Enzymology 185:537)
reviews several transformation protocols. Such techniques may
utilize many different conditions including, but not limited to,
various art-recognized vectors, numerous cell lines, and diverse
culture conditions.
[0132] A wide variety of host cells may be transfected. These cells
may be either of eukaryotic or of prokaryotic origin. The cells
will often be immortalized eukaryotic cells, and in particular,
mammalian cells, for example monkey kidney cells (COS), Chinese
hamster ovary cells (CHO), HeLa cells, baby hamster kidney cells
(BHK), human embryonic kidney cells (HEK293), leukocytes, myelomas,
and embryonic stem cells. The cells may also be non mammalian cells
including bacterial, fungi, yeast and insect cells, including, but
not limited to, for example Esherichia coli, Bacillus subtilus,
Aspergilliis species, Saccharomyces cerevisiae, and Pichia
pastoris. All cells may be grown in culture trays medium under
appropriate conditions or in a synergistic host. The most desirable
cells will be mammalian cells capable of culture.
[0133] The secreted POI bound to the cell surface capture molecule
may be detected and isolated by various techniques known in the
art. Cultures cells displaying the secreted POI may be contacted
with (a) molecule(s) capable of directly or indirectly binding the
secreted POI wherein such detection molecule(s) may contain a
detection label, such as, for example, a chromogenic, fluorogenic,
colored, fluorescent, or magnetic label. The label bound to the
detection molecule may be detected and the cell isolated using
various methods. Most preferably, within a cell population the
label will be detected and the cell isolated utilizing flow
cytometry.
[0134] Alternatively, the detection molecule may be used for the
direct isolation of cells displaying the POI. This may be
accomplished by conjugation of the detection molecule to a culture
plate, paramagnetic molecules, or any other particle or solid
support. In addition, displayed POI may be detected directly by a
property of the detection molecule or the POI.
[0135] In one embodiment, two detection molecules that bind each
other and are differentially labeled are used to detect a displayed
secreted POI that blocks that interaction. If a cells displays a
secreted POI that binds the first detection molecule and blocks the
interaction between the first and second detection molecule, that
cell may be isolated based on the presence of only the first
detection molecule on its surface. On the other hand, if a cell
displays a secreted POI that binds the first detection molecule but
does not block the interaction between the first and second
detection molecule, that cell may be isolated based on the presence
of both detection molecules on its surface. For example, antibody
producing cells expressing antibodies that specifically block, or
do not block, the formation of a receptor-ligand complex may be
identified. If the detection molecules are a receptor and its
ligand which are differentially labeled, then an antibody producing
cell that expresses antibodies that block the receptor-ligand
complex from forming may be detected by the presence of one label
on its surface, whereas an antibody producing cell that expresses
antibodies that do not block the receptor-ligand complex from
forming may be detected by the presence of both labels on its
surface.
[0136] In any of the embodiments and with regards to isolating
expressing cells from non-expressing cells or lesser expressing
cells, one of the principal difficulties, when the POI is a
secreted protein, is diffusion of POI between neighboring cells.
Therefore, it is critical that any system that is designed to
capture the secreted POI on the cell surface must prevent the
diffusion of the POI from the expressing cell to a neighboring cell
and its adherence to that cell. If diffusion is allowed to occur,
and neighboring cells become decorated with the secreted POI, then
separation of cells based upon the degree of POI decoration will
fail to discriminate high expressing cells from cells with low
expression levels, and may fail to effectively isolate expressing
from non-expressing cells.
[0137] Therefore one aspect of this invention is to block the
diffusion of the secreted POI between neighboring cells. This may
be accomplished by the addition of a blocking molecule that binds
either the cell surface capture molecule or the POI and prevents
the binding of the secreted POI to the cell surface capture
molecule. In this aspect, the detection molecules do not bind the
blocking molecule. For example, if the cell surface receptor is the
hFc gamma RI and the secreted POI possesses the human IgG Fc
fragment, then diffusion of the secreted POI between neighboring
cells may be blocked by the addition of exogenous rat IgG to the
culture media. Detection of cells displaying secreted POI, and not
bound rat IgG, is achieved by use of antibodies specific for human
IgG Fc that do not recognize rat IgG.
[0138] In another aspect of this invention, binding of the secreted
POI between neighboring cells is reduced by increasing the
viscosity of the media.
[0139] In another aspect of this invention, the secreted POI is not
allowed to accumulate in the media. This may be accomplished by
regulating the expression of the secreted POI and/or the cell
surface capture molecule such that brief expression of the POI
results in sufficient POI to bind the cell surface capture molecule
but insufficient amounts for diffusion.
[0140] In yet another aspect of this invention, cells may be
removed from the media containing accumulated POI, the POI bound to
the cells is stripped off, and POI expression is allowed to
continue for a limited period of time such that secreted POI does
not accumulate in the media. Proteins may be stripped by methods
known in the art, for example, washing cells with low pH
buffer.
[0141] According to this invention, those cells in a cell
population that bind the most detection molecules also express the
most secreted POI. In fact, the more POI that an individual cell
secretes, the more POI is displayed on the cell surface. This
correlation between the amount of surface-displayed POI and the
expression level of the POI in that cell allows one to rapidly
identify cells with a desired relative expression level from a
population of cells.
[0142] In one embodiment, a DNA library may be used to express
secreted protein which may be displayed on the cell surface by the
cell surface capture molecule. For example, a library of DNA may
also be generated from the coding regions of the antibody variable
domains from B-cells isolated from immunized animals. The DNA
library may then be expressed in a cell that expresses a cell
surface capture molecule specific for antibodies such that clones
of desired specificity, isotype, or avidity may be identified and
isolated by the method of the invention.
[0143] In another embodiment, transgenic mammals may be created
which express a particular cell surface capture molecule in one or
more cell types. The cells from such transgenic mammals may then be
screened directly for the production of a POI. For example, it may
be desirable to express a cell surface capture molecule, specific
for antibodies, in plasma cells. Accordingly, plasma cells from
immunized mice may be harvested and those cells producing
antibodies specific to the desired antigen may be isolated by the
method of the invention.
[0144] In a further embodiment of the invention, antibody
production is measured through the use of a CHO cell line that
expresses the human Fc gamma R1 receptor (Fc.gamma.RI) which binds
the particular antibody that is the POI.
EXAMPLE 1
[0145] Construction of pTE084:
[0146] pTE084 was constructed by ligating the 1,436 bp Xba I
fragment from pCAE100 that encodes the human Fc.gamma.RI
(hFc.gamma.RI; GenBank accession number M21091) into the Xba I site
of pRG821. The orientation of hFc.gamma.RI in desirable plasmids
resulting from the ligation was examined by restriction mapping
with Not I, Pst I, Eco RI, and Stu I. pTE084 was designed for the
high level expression of hFc.gamma.RI, the high affinity cell
surface receptor for the Fc domain of human IgG. It contains two
independent expression cassettes. One cassette is a hFc.gamma.RI
gene driven by the CMV-MIE promoter, and the second cassette is the
neomycin phosphotransferase 11 (npt) gene, which confers resistance
to G418, driven by the SV40 late promoter.
[0147] Construction of a CHO K1 Derivative That Expresses
hFc.gamma.RI:
[0148] CHO K1 cells (4.times.10.sup.6) were transfected with pTE084
using Lipofectamine.TM. (Life Technologies; Rockville, Md.)
following manufacturer's suggestions. The cells were placed in the
culture medium (10% fetal bovine serum, 90% Ham's F-12, 2 mM
L-glutamine; all reagents were from Life Technologies, Rockville,
Md.) containing 500 ug/ml G418 (Life Technologies) for 15 days. The
cells that survived G418 selection were trypsinized, pooled, and
stained with FITC-conjugated human IgG, Fc fragment (FITC-hFc;
Jackson ImmunoResearch Laboratories, West Grove, Pa.). Briefly, the
cells grown on 10 cm culture plates were washed once with
Dulbecco's phosphate-buffered saline (PBS) without calcium chloride
and magnesium chloride (Life Technologies). Three mls of 0.25%
trypsin (Life Technologies) was added to each plate. The plates
were swirled until the cells detached from the plate. 10 ml culture
medium was immediately added to each plate of the detached cells.
The cells were then collected by centrifugation at 1,000.times. g
for 4 minutes. After removal of supernatant, the cells were
resuspended in 4 ml of 2 ug/ml FITC-hFc diluted in culture medium.
The cells were then placed on a platform shaker and stained for one
hour at room temperature. To remove unbound FITC-hFc, the cells
were washed twice with 20 ml PBS. The degree of FITC-hFc label on
the cells was measured by flow cytometry on a Moflo cell sorter
(Cytomation; Fort Collins, Colo.). The FITC-hFc did not stain
mock-transfected parental CHO K1 cells (FIGS. 2A and 2B) but gave
rise to a distribution of fluorescence in the G418-resistant,
pTE084-transfected pool (FIG. 2C). The top 1% most fluorescent
cells from the selected pool were placed into 96-well plates at 1
cell/well by flow cytometry. Nine days later, 88 cell clones in the
96-well plates were expanded into 24-well plates. After 3 days, the
cells in individual wells were washed once with 1 ml PBS, stained
with 0.5 ml of 2 ug/ml FITC-hFc for 1 hour, washed twice with 1 ml
PBS and examined for cell surface staining under a fluorescent
microscope. The thirty three most fluorescent clones were chosen,
expanded, then screened by flow cytometry. The FITC-hFc staining of
one such clone, RGC3, was shown in FIG. 2D.
[0149] Diffusion of Secreted Protein Between Expressing Cells and
Non-Expressing Cells Among Cells was Blocked by Adding IgG:
[0150] As all cells in a hFc.gamma.RI clonal cell line express a
cell surface hFc.gamma.RI, they all possess the ability to bind IgG
or fusion proteins consisting of the Fc domain of IgG. Because
hFc.gamma.RI binds IgG from a variety of species (van de Winkel and
Anderson, 1991), a panel of animal IgGs was tested for the ability
to block the binding of a protein containing a human IgG1 (hIgG1)
Fc tag (4SC622) to hFc.gamma.RI-expressing cells. 4SC622 is a
chimeric molecule consisting of IL-2R.gamma. extracellular domain
fused to the hIL-4R.gamma. extracellular domain which is then fused
to the hIG-1Fc domain. In this experiment, cultures of RGC1, a
hFc.gamma.RI-expressing cell line selected from CHO K1 cells that
have been stably transfected with pTE084, were incubated with 1
.mu.g/ml 4SC622 for 18 hours in the presence or absence of 1 mg/ml
IgG from different species in a 37.degree. C. tissue culture
incubator. Cell surface binding of 4SC622 was determined by flow
cytometry after washed cells were stained with
phycoerythrin-conjugated mouse IgG1 monoclonal AG184 (PE-AG184)
specific for the hIL-2R.gamma. component of 4SC622 (BD PharMingen;
San Diego, Calif.), following procedures outlined for cell staining
with FITC-hFc. FIG. 3 shows that hIgG completely blocked 4SC622
from binding to the hFc.gamma.RI expressed on the surface of RGC1.
Rat, rabbit and canine-derived IgG also effectively blocked binding
whereas bovine and ovine-derived IgG did not block. The ability of
exogenously added rat IgG to block the binding of an exogenously
added hIgG1 Fc-tagged protein (4SC622) to cell surface hFc.gamma.RI
(FIG. 4) suggests that rat IgG can also block transfer between
cells expressing a hIgG1 Fc-tagged protein at different levels. To
test this, two cell lines that can be distinguished by the presence
or absence of the green fluorescent protein (EGFP) were generated
from RGC1. Briefly, to mark RGC1 cells with EGFP, 2.times.10.sup.6
RGC1 cells were co-transfected with 0.5 .mu.g PTE073 which encodes
a hygromycin B phosphotransferase gene driven by phosphoglycerate
kinase promoter, and 5 .mu.g pRG816-EGFP which encodes EGFP gene
driven by CMV-MIE promoter. The transfected cells were selected
with 200 ug/ml hygromycin B (Sigma; St. Louis, Mo.) for two weeks.
Green fluorescent cells were isolated by flow cytometry. One EGFP
and hFc.gamma.RI-expressing clone, RGC2, was used in cell mixing
experiments. The other cell line used in these experiments, RGC4,
was generated by stable transfection of RGC1 with plasmid
pEE14.1-622. pEE14.1-622 is a plasmid in which expression of 4SC622
is driven by the CMV-MIE promoter and includes a glutamine
synthetase minigene, which confers resistance to the analog
methionine sulfoximine (MSX), and allows for selection of stable
integration events. RGC4 cells express hFc.gamma.RI on the cell
surface and secrete the hIgG1 Fc-tagged protein 4SC622. One plate
of mixed cells comprised of 50% RGC2 and 50% RGC4 cells was
incubated with 1 mg/ml rat IgG for 18 hours prior to staining with
PE-AG184 then examined by flow cytometry. FIG. 5A shows the EGFP
flourescence of RGC2 cells and FIG. 5B shows that RGC2 cells also
bind exogenously added 4SC622 (1 .mu.g/ml) as indicated by an
increase in PE-AG184 fluorescence. FIG. 5C shows that RGC4 did not
fluoresce in the EGFP gate. Significantly, exogenously added rat
IgG did not reduce the percentage of RGC4 cells that stained
positive for cell surface 4SC622 (FIG. 5D) suggesting that the
binding of 4SC622 to hFc.gamma.RI occurred while the proteins were
in transit to the cell surface. When RGC2 and RGC4 cells were mixed
(FIG. 5E), the 4SC622 protein secreted from RGC4 cells accumulated
in the medium and bound most of the RGC2 cells. However, the
addition of 1 mg/ml rat IgG significantly reduced the percentage of
RGC2 cells that bound 4SC622 (FIG. 5F) demonstrating that rat IgG
blocked the transfer of secreted hIgG1 Fc-tagged protein from
expressing cells to non-expressing cells.
EXAMPLE 2
[0151] Cell Surface Fluorescence Correlates with the Expression
Level of 4SC622:
[0152] RGC1 cells (4.times.10.sup.6) were transfected with
pEE14.1-622 and a pool of stable transfectants was obtained after
selection for 2 weeks in medium comprised of 10% dialyzed fetal
bovine serum, 90% glutamine-free Dulbecco's modified eagle's
medium, 1.times. GS supplement, and 25 uM MSX (All reagents were
from JRH Biosciences, Lenexa, Kans.). Rat IgG was added to the
culture medium to 1 mg/ml 18 hours prior to immunostaining. The
cells were trypsinized, washed with PBS, and stained with 1.5 ug/ml
of a polyclonal FITC-conjugated anti-human IgG (H+L)
F(ab').sub.2fragment (Jackson ImmunoResearch Laboratories) for one
hour at room temperature following procedures as described for
FITC-hFc staining in Example 1. Cell staining was then analyzed by
flow cytometry. The distribution of fluorescence suggested that the
selected pool contained cells with a wide range of 4SC622
expression levels (FIG. 6). Cells in the top 3% (R3 bracket), 7-11%
(R5 bracket), and 1519% (R7 bracket) with respect to their
immunofluorescence were sorted into three distinct pools and
expanded for 9 days. Average 4SC622 production per cell for the
pools was determined by measuring cell numbers and 4SC622 levels in
the media after 3 days growth by an immuno-based Pandex assay
(Idexx; Westbrook, Me.) following the manufacturer's
recommendations. In the Pandex assay, fluoricon polystyrene assay
particles coated with goat antihuman IgG, .gamma.-chain specific
antibody (Sigma) were used to capture 4SC622 from the medium, and a
FITC-conjugated goat anti-human IgG, Fc specific (Sigma) was used
to detect bead-bound 4SC622. Known amounts of purified 4SC622 were
included in the assay for calibration. Cells in the top 3%, 7-11%,
and 15-19% pool were found to produce 4SC622 at 1.42, 0.36, and
0.22 pg/cell/day, respectively. Thus, there was a correlation
between cell surface 4SC622 staining and specific protein
production. This result suggests that individual cells that express
4SC622 at high levels may be obtained by isolating cells that were
stained brightest by the polyclonal FITC-conjugated anti-human IgG
(H+L) F(ab').sub.2 fragment.
EXAMPLE 3
[0153] Isolation of Expression Clones in RGC1:
[0154] IL-4 Trap.
[0155] To directly demonstrate the efficiency in generating clonal
cell lines with high level secreted protein production by our
methodology, clonal 4SC622 producing cell lines were generated from
RGC1. RGC1 cells (4.times.10.sup.6) were transfected with
pEE14.1622, and selected for two weeks with 25 .mu.M MSX to obtain
a pool of stable transfectants. MSX-resistant cells were pooled and
incubated with 1 mg/ml human IgG for 18 hours, prior to staining
with PE-AG184. Six cells from the top 5% gate, as determined by
flow cytometry analysis of cell surface 4SC622 staining, were
isolated and expanded. 4SC622 production from the six clonal lines
was determined and compared to 4SC622 production from clones
obtained by hand-picking selected colonies followed by dilution
cloning and amplification. One RGC1-derived clone, RGC4, produced
4SC622 at 12 pg/cell/day (FIG. 7). This level is similar to that of
the best 4SC622 producer isolated by hand-picking and analyzing
2,700 clones. Thus, compared with hand-picking colonies, the
methodology outlined in this invention proves to be far more
efficient in the screening and cloning of high producers.
[0156] VEGF Trp.
[0157] Plasmids pTE080 and pTE081 encode the genes for VEGF Traps,
hVEGFR1R2 and hVEGF-R1R3. hVEGF-R1R2 is a chimeric molecule
consisting of the first Ig domain of hVEGFR1 fused to the second Ig
domain of hVEGFR2 which is then fused to the hIg1FC domain.
hVEGFR1R3 is a chimeric molecule consisting of the first Ig domain
of hVEGFR1 fused to the second Ig domain of hVEGFR3 which is then
fused to the hIg1FC domain. In these plasmids, the gene for the
VEGF Trap is driven by the CMV-MIE promoter and a glutamine
synthetase minigene, which confers resistance to MSX, is expressed
for selection of stable integration events. RGC1 cells were
transfected with either of these plasmids and grown in medium
containing 25 .mu.M MSX for 2 weeks to select for cells in which
the plasmid has stably integrated. MSX-resistant cells were
incubated with 0.1 ug/ml Ig2a and mouse IgG3 for 18 hours prior to
staining with 1.5 ug/ml polyclonal FITC-conjugated antihuman IgG
(H+L) F(ab').sub.2fragment. Cell were stained for 1 hour then
washed twice with PBS prior to flow cytometry. Single cells were
sorted into 96-well tissue culture plates from the pool of cells
whose fluorescence was among the highest 1%. The cells in
individual wells were expanded and their productivities were
determined by Pandex assays. FIG. 7 shows that RGC-derived clones
expressing both hVEGFR1R2 and hVEGFR1R3 had higher specific
productivities and were isolated by screening fewer clones as
compared to the highest-expressing hand-picked MSX-resistant
colonies.
EXAMPLE 4
[0158] Cell Surface-Bound hIgG1 Fc-Tagged Protein is Internalized
by RGC1:
[0159] hFc.gamma.RI is known to induce internalization of its cell
surface-bound ligand. To analyze whether RGC1 cells could
internalize cell surface-bound 4SC622, 1 ug/ml 4SC622 was added to
RGC1 cells for 1 hour and then the cells were immediately processed
for 4SC622 immunostaining with PE-AG184 and flow cytometry
analysis. 93% of the cells stained positive for cell surface 4SC622
(FIG. 8B). Alternatively, 1 ug/ml 4SC622 was added to RGC1 cells
for 1 hour, then the cells were washed and incubated in culture
medium without 4SC622 with PE-AG184 for 18 hours. Flow cytometry
analysis following immunostaining for 4SC622 showed that 9% of the
cells retained 4SC622 on the cell surface (FIG. 8C). To further
characterize the loss of surface-bound 4SC622, purified 4SC622
protein was added to the media of RGC1 and parental CHO K1 cells,
then levels of 4SC622 in the media were measured over time. FIG. 9
shows that 4SC622, added to 2 .mu.g/ml to the culture media in a 10
cm plate, was significantly lower in RGC1 conditioned medium after
3 days incubation as compared to the CHO K1 control. These results
show that the concentration of 4SC622 in the culture medium is
reduced by the presence of hFc.gamma.RI on the cell surface. The
results suggest that the depletion of 4SC622 from the media was the
result of hFc.gamma.RI-4SC622 complex internalization. This
internalization of receptor-ligand complexes may facilitate the
effective removal of all 4SC622 from non-expressing cells in the
presence of blocking IgG during the 18 hour blocking step.
EXAMPLE 5
[0160] Construction of CHO K1 Cell Lines with Inducible
hFc.gamma.RI Expression:
[0161] Flow cytometry-based autologous secretion trap methods that
utilize the hFc.gamma.RI allow rapid isolation of high expression
clones. However, if hFc.gamma.RI mediates turnover of Fc-tagged
proteins, then the realized production of the secreted protein by
engineered hFc.gamma.RI expressing cells would be higher if
hFc.gamma.RI expression could be inhibited during the production
period. To this end, a CHO K1 cell line in which the expression of
hFc.gamma.RI is induced by tetracycline, or the analog doxycycline,
was constructed. In this system, CHO K1 cells were first engineered
to express the tetracycline repressor protein (TetR) and
hFc.gamma.RI was placed under transcriptional control of a promoter
whose activity was regulated by TetR. Two tandem TetR operators
(TetO) were placed immediately downstream of the CMV-MIE
promoter/enhancer in pTE084 to generate pTE158 (FIG. 10).
Transcription of hFc.gamma.RI from the CMV-MIE promoter in pTE158
was blocked by TetR in the absence of tetracycline or some other
suitable inducer. In the presence of inducer TetR protein was
incapable of binding TetO and transcription of hFc.gamma.RI
occurs.
[0162] CHO K1 cells were transfected with pcDNA6/TR, a plasmid that
confers resistance to blasticidin in which expression of TetR
originates from the CMV-MIE promoter (Invitrogen; Carlsbad, Calif.)
After two weeks of selection with 2.5 ug/ml blasticidin
(Invitrogen), the stable transfectants were pooled. This pool was
then transfected with pTE158, a plasmid that confers resistance to
G418 in which the expression of hFc.gamma.RI is dependent on a
CMV43 MIE/TetO hybrid promoter. The cells consecutively transfected
with pcDNA6/TR and pTE158 were selected with 400 ug/ml G418 and 2.5
ug/ml blasticidin for 12 days then pooled. The pool was induced for
two days by the addition of 1 .mu.g/ml doxycycline then stained
with FITC-hFc to identify cells that express hFc.gamma.RI. The top
5% of cells expressing hFc.gamma.RI were collected as a pool,
expanded for 6 days in the absence of doxycycline, and were again
stained with FITC-hFc for the presence of hFc.gamma.RI. Cells that
did not stain for hFc.gamma.RI were collected and expanded in
culture medium containing 1 .mu.g/ml of doxycycline for three days.
The pool was then stained for the presence of hFc.gamma.RI and were
isolated by flow cytometry. Cells that expressed the highest levels
of hFc.gamma.RI (top 1%) were sorted onto 96 well plates at one
cell per well. These cells presumably contained cell that had low
non-induced expression levels of FcR1 and high inducible levels of
FcR1. After expansion, the induction of hFc.gamma.RI by doxycycline
in 20 clones was confirmed by immunostaining with FITC-hFc and flow
cytometry. One clone was chosen for further characterization and
was named RGC10. FIG. 11 shows that in the absence of doxycycline
RGC10 did not express detectable levels of hFc.gamma.RI, whereas
high levels of hFc.gamma.RI were observed in cells that were
induced with 1 .mu.g/ml of doxycycline for three days. The mean
fluorescence of RGC10 cells increased by more than 1,000 fold after
induction by doxycycline.
EXAMPLE 6
[0163] Isolation of 4SC622-Producing Cell Lines from RGC10:
[0164] RGC10 cells were transfected with pEE14.1-622, and
MSX-resistant cells were pooled after selection with 25 .mu.M MSX
for two weeks. Expression of hFc.gamma.RI was induced by the
addition of 1 .mu.g/ml of doxycycline to the culture medium for
three days. One mg/ml rat IgG was added to the culture medium
containing doxycycline 18 hours prior to staining with polyclonal
FITC-conjugated anti-human IgG (H+L) F(ab').sub.2fragment and
analysis by flow cytometry. Cells that expressed the highest levels
of 4SC622 (top 1%) were sorted into 96 well plates at 1 cell per
well (FIG. 12C). FIG. 12B shows that without induction of
hFc.gamma.RI expression by doxycycline, staining with polyclonal
FITC-conjugated anti-human IgG (H+L) F(ab').sub.2 fragment fails to
detect cell surface bound 4SC622. Sixty clones were expanded in the
absence of doxycycline. FIG. 13 shows the specific productivity of
the 13 highest producers as determined by Pandex assay. The
specific productivity of clone 1C2 was 17.8 pg/cell/day,
significantly better than the 12 pg/cell/day observed for the best
4SC622 cell line previously isolated using the unregulated
hFc.gamma.RI cell line RGC1.
EXAMPLE 7
[0165] Sp2/0 Myeloma Cells can be Engineered to Express a Cell
Surface Capture Protein:
[0166] In this example, the Sp2/0-Ag14 myeloma cell line was
engineered to stably express hFc.gamma.RI to demonstrate that the
autologous secretion trap method was applicable to cell lines other
than CHO. The gene for hFc.gamma.RI was introduced into the myeloma
cell by retroviral infection. The plasmid pLXRN (Clontech; Palo
Alto, Calif.), a retroviral DNA vector wherein a gene of interest
may be expressed from the upstream Moloney murine sarcoma virus
long terminal repeat (MoMuSV LTR) promoter, was used to generate
retrovirus encoding the hFc.gamma.RI gene. The 1,363 bp Xho I
fragment from pTE084, encoding the human Fc.gamma.RI gene, was
cloned into the Xho I site of pLXRN. A plasmid in which
hFc.gamma.RI cDNA expression was dependent on the MoMuSV LTR was
chosen and named pTE255 (FIG. 14).
[0167] Pantropic retrovirus for the expression of hFc.gamma.RI was
generated essentially following the manufacturer's guidelines. The
packaging cell line GP-293, a HEK 293-based cell line that stably
expresses the viral gag and pol proteins (Clontech; Palo Alto,
Calif.), was co-transfected with 10 .mu.g each of pVSV-G and
pTE255. The plasmid pVSV-G allows expression of the viral envelope
protein VSV-G that confers broad host range upon the infective
particles.
[0168] Construction of Sp2-hFc.gamma.RI-4:
[0169] The pantropic hFc.gamma.RI retrovirus was used to infect
1.times.10.sup.7 Sp2/0Ag14 myeloma cells (American Type Culture
Collection; Manassas, Va.) at a multiplicity of about 10 infective
particles per cell. Three days after infection, cells were stained
for 1 hour then washed twice with PBS prior to analysis by flow
cytometry. Those cells expressing hFc.gamma.RI, as indicated by
bound FITC-hFc, were collected as a pool by flow cytometry. The
pool was expanded for 13 days then again stained with FITC-hFc and
cells expressing hFc.gamma.RI were collected as a pool by flow
cytometry. These sorted cells were cultured in 10% fetal bovine
serum 90% Dulbecco's Modified Eagle's Medium (D-MEM) with 4.5 g/l
glucose and 4 mM glutamine for 3 weeks, stained with FITC-hFc, and
the cells with mean fluorescence in the top 1% of the population
were cloned by single cell sorting. After expansion, 24 clones were
examined by flow cytometry for expression of hFc.gamma.RI, as
described above, and one clone, Sp2-hFc.gamma.RI-4, was chosen for
additional characterization (FIG. 15).
[0170] Isolation of Sp2-hFc.gamma.RI-4 Cells Expressing 4SC622
Protein:
[0171] Sp2-hFc.gamma.RI-4 cells (1.times.10.sup.7)were transfected
with pTE209 (FIG. 16), a plasmid that allows constitutive
expression of 4SC622 from the CMV-MIE promoter and confers
resistance to hygromycin. The transfected cells were placed in
medium containing 10% FCS, 90% D-MEM and 400 .mu.g/ml hygromycin
for 14 days. Hygromycin-resistant cells were incubated with 1 mg/ml
rabbit IgG for eighteen hours prior to staining with polyclonal
FITC-conjugated anti-human IgG (H+L) F (ab').sub.2fragment. Cells
were stained for 1 hour then washed twice with PBS prior to
analysis by flow cytometry. Labeled cells were collected as a pool
by flow cytometry then cultured for 5 days and sorted as described
above. Cells from the expanded pool that bound the most polyclonal
FITC-conjugated anti-human IgG (H+L) F (ab').sub.2fragment, top 1%
population, were then cloned by single cell sorting (FIG. 17).
Production of 4SC622 from ten clones was analyzed by ELISA and all
10 clones were found to express 4SC622; clone 5H11 produced 4SC622
at 0.5 pg per cell per day. These data showed that clones secreting
4SC622 were efficiently isolated by the autologous secretion trap
method from a heterogeneous pool of cells derived from stable
transfection of Sp2-hFc.gamma.RI-4 cells with pTE209.
[0172] To confirm that 4SC622 was autologously displayed on the
surface of myeloma cells expressing both 4SC622 and hFc.gamma.RI,
clone 5H11 was incubated with 1 mg/ml rabbit IgG for 18 hours then
stained with FITC-conjugated anti-human IgG (H+L)
F(ab').sub.2fragment and found to display cell surface 4SC622.
Secreted protein was displayed under conditions in which
cross-feeding was blocked by rabbit IgG, demonstrating the
autologous display of 4SC622. These data indicated that the
autologous secretion trap method described above was not limited to
CHO cells and may be extended to myeloma and other cell types as
well.
EXAMPLE 8
[0173] A Protein G Chimeric Protein can Function as a Cell Surface
Capture Protein:
[0174] To demonstrate the application of the autologous secretion
trap method to a cell surface capture protein other than
hFc.gamma.RI, a cell line expressing Protein G was constructed.
Protein G, from the Streptococcus strain G148, binds to all human
and mouse IgG subclasses, and as such has utility for the isolation
of recombinant cells expressing antibodies or IgG Fc fusion
proteins. To demonstrate that the Protein G IgG Fc binding domain
could be used as a cell surface capture protein capable of binding
to all human and mouse IgG subclasses, we constructed a CHO line
expressing a chimeric protein comprised of the Fc binding domain of
Protein G fused to the hFc.gamma.RI transmembrane and intracellular
domain (FIG. 18). The Fc binding domain of Protein G contains three
homologous repeats of 55 amino acids long (Guss et al., (1986) EMBO
5:1567 and Sjobring et al., (1991) J. Biol. Chem. 266:399) and each
repeat is capable of binding one IgG Fc. To improve the expression
of this chimeric protein in CHO cells, we constructed a synthetic
DNA in which the signal sequence from the mouse ROR1 gene was fused
to the Fc binding domain, amino acids 303 to 497, of Protein G
(accession # X06173). This synthetic DNA was generated by a
combination of oligonucleotide annealing, gap filling, and PCR
amplification. The synthetic DNA was then fused, by PCR, to DNA
encoding the transmembrane and intracellular domains, amino acids
279 to 374, of hFc.gamma.RI (accession M21091). The resultant DNA
encoding the Protein G/hFc.gamma.RI chimeric protein was cloned
into pTE158 downstream of the CMV-MIE promoter, replacing the gene
encoding hFc.gamma.RI, to yield the plasmid pTE300 (FIG. 19).
[0175] A CHO K1 cell line adapted to grow in serum-free medium,
RGC14, was transfected with pTE300, and after three days 400
.mu.g/ml G418 was added to the culture medium to select for stable
integration of pTE300. Two weeks after the start of selection, the
cells were stained with FITC-hFc to identify cells that expressed
hFc.gamma.RI. These cells were analyzed by flow cytometry and cells
expressing hFc.gamma.RI were collected as a pool (FIG. 20). The
cells were expanded for 10 days and the population of cells
expressing hFc.gamma.RI was again isolated by flow cytometry. The
cells were again expanded, stained with FITC-hFc, and single cells
expressing high levels of the Protein G/hFc.gamma.RI chimeric
protein were isolated by flow cytometry. Single cells that stained
positive for FITC-hFc binding were sorted into medium composed of
10% fetal bovine serum, 90% Ham's F12, and 400 .mu.g/ml G418. After
two weeks incubation, 48 clones were examined for binding to bovine
IgG present in the culture medium by staining with FITC-conjugated
anti-bovine IgG F(ab').sub.2 fragment (Jackson ImmunoResearch
Laboratories, West Grove, Pa.). One clone, RGC18 that stained
positive with this antibody was chosen for further
characterization.
[0176] Isolation of Expression Clones in RGC18:
[0177] RGC18 cells (6.times.10.sup.6) were transfected with pTE209
and selected for integration of the plasmid by growth in 400
.mu.g/ml hygromycin for 18 days. Hygromycin-resistant cells were
incubated with 1 mg/ml rabbit IgG for eighteen hours prior to
staining with polyclonal FITC-conjugated anti-human IgG (H+L) F
(ab').sub.2fragment. Cells were stained for 1 hour then washed
twice with PBS prior to analysis by flow cytometry (FIG. 21). The
most fluorescent cells (top 5%) were isolated by single cell
sorting and expanded for 3 weeks. Ten clones were examined for
4SC622 secretion. All clones tested secreted 4SC622 at high level,
and the best clone, RGC19, had a specific productivity of 6.4
pg/cell day. This result demonstrated that 4SC622-expressing cells
were efficiently isolated from a heterogeneous pool of cells
derived from stable transfection of RGC18 with pTE209 by the
autologous secretion trap method. Furthermore, these data clearly
demonstrated that a fragment of Protein G could be engineered to
include a signal sequence and transmembrane domain, and function as
a cell surface capture protein.
[0178] To confirm that 4SC622 was autologously displayed on the
surface of RGC19 cells expressing both Protein G/hFc.gamma.RI
chimeric protein and 4SC622, RGC19 was incubated with 1 mg/ml
rabbit IgG for 18 hours then stained with FITC-conjugated
anti-human IgG (H+L) F(ab').sub.2fragment and analyzed by flow
cytometry. RGC19 cells were found to possess cell surface 4SC622
under these conditions in which cross-feeding was blocked by rabbit
IgG, suggesting autologous display of 4SC622 (FIG. 22). Rabbit IgG
effectively blocked binding of exogenous 4SC622 protein to RGC18
cells, but did not block display of 4SC622 on the cell surface of
cells expressing 4SC622. These data demonstrated that the
properties of the Protein G/hFc.gamma.RI chimeric protein were
similar to those of hFc.gamma.RI as a cell surface capture protein,
and suggested that the autologous secretion trap method can employ
other proteins as cell surface capture proteins.
EXAMPLE 9
[0179] Isolation of Antibody-Producing Cells from RGC10:
[0180] To demonstrate the utility of the autologous secretion trap
method for the isolation of CHO cell lines that express recombinant
antibodies we cloned the DNA encoding variable light and variable
heavy genes from the KD5 hybridoma. KD5 is a hybridoma that
expresses a monoclonal antibody specific for the human Tie-2
receptor.
[0181] The mouse IgG constant region gene sequences were cloned
from 500 ng of mouse spleen polyA+ RNA (Clontech, Palo Alto,
Calif.). Single stranded cDNA was synthesized using SuperScript
First-Strand Synthesis System for RT-PCR, primed with 50 ng of
random hexamers (Invitrogen Life Technolgies, Carlsbad, Calif.).
The mouse kappa light constant DNA sequence (accession # Z37499)
was amplified from this cDNA by PCR using the primers 5' mCLK1
(Z37499) (5'-CGGGCTGATGCTGCACCAACTGTATCCATCTTC-3') and 3' mCLK1
(Z37499) (5'-ACACTCTCCCCTGTTGAAGCTCTTGACAATGGG-3'). The mouse IgG2a
constant region DNA sequence (accession # AJ294738) was also
amplified from this cDNA by PCR using the primers 5'
mCH2a(AJ294738) (5'-GCCAAAACAACAGCCCCATCGGTCTATCCAC-3') and 3'
mCH2a(AJ294738) (5'-TCATTTACCCGGAGTCCGGGAGAAGCTCTTAGTCG-3'). The
PCR products were cloned into pCR2.1-TOPO using TOPO TA Cloning kit
(Invitrogen Life Technolgies, Carlsbad, Calif.) and the sequence of
the constant regions were verified.
[0182] The KD5 variable region genes were amplified by RT-PCR from
KD5 hybridoma mRNA and cloned into pCR2.1-TOPO using the heavy and
light chain variable region primer mixes from Amersham-Pharmacia
Biotech (Piscataway, N.J.). The variable heavy chain gene was PCR
amplified using the pCR2.1-TOPO cloned variable region as template
with the primers 5' BspMI/KD5VH N-term
(5'-GAGAGTACCTGCGTCATGCAGATGTGAAACTGCAGGAGTCTGGCCCT-3'- ) and 3'
BspMI/KD5VH C-term (5'-GAGAGACCTGCGTCAGCTGAGGAGACGGTGACCGTGGT-3')-
, digested with BspMI and ligated to the BsaI-digested IgG2a
constant heavy gene PCR fragment amplified with the primers 5'
BsaI/CH2a N-term (5'-GAGAGGGTCTCACAGCCAAAACAACAGCCCCATCG-3') and 3'
BsaI/CH2a C-term
(5'-GAGAGGGTCTCCGGCCGCTCATTTACCCGGAGTCCGGGAGAA-3'). This fragment
was then ligated into the BspMI and NotI sites of pRG882. The
resulting plasmid, pTE317, was capable of expressing the KD5
recombinant heavy chain gene, fused to the mROR1 signal sequence,
from the CMV-MIE promoter. The variable light chain gene was PCR
amplified using the pCR2.1-TOPO cloned variable region as template
with the primers 5' BsmBI/KD5VL N-term
(5'-GAGAGCGTCTCATGCAGACATCCAGATGACCCAGTCTCCA-3') and 3' BsmBI/KD5VL
C-term (5'-GAGAGCGTCTCACAGCCCGTTTTATTTCCAGCTTGGTCCC-3'), digested
with BsmBI and ligated to the BsaI-digested kappa constant light
gene PCR fragment amplified with the primers 5' BsaI/CLK N-term
(5'-GAGAGGGTCTCAGCTGATGCTGCACCMCTGTATCC-3') and 3' BsaI/CLK C-term
(5'-GAGAGGGTCTCAGGCCGCTCMCACTCTCCCCTGTTGAAGCTCTTGAC-3'). This
fragment was then ligated into the BspMI and NotI sites of pRG882.
The resulting plasmid, pTE316, was capable of expressing the KD5
recombinant light chain gene, fused to the mROR1 signal sequence,
from the CMV-MIE promoter.
[0183] The 1450 bp EcoRI-NotI fragment from pTE317, encoding the
KD5 heavy chain gene, was cloned into the EcoRI and NotI sites of
pRG980, a vector that confers resistance to hygromycin and allows
expression of recombinant genes for the UbC promoter, to yield
plasmid pTE322. Similarly, the 750 bp EcoRI-NotI fragment from
pTE316, encoding the KD5 light chain gene, was cloned into the
EcoRI and NotI sites of pRG985, a vector that confers resistance to
puromycin and allows expression of recombinant genes for the UbC
promoter, to yield plasmid pTE324.
[0184] RGC10 cells (5.times.10.sup.6) were transfected with 3 .mu.g
pTE322 and 3 .mu.g pTE322 and selected for integration of the
plasmids by growth in F12 medium supplemented with 10% fetal calf
serum with 20 .mu.g puromycin and 400 .mu.g/ml hygromycin for 14
days. Expression of hFc.gamma.RI was induced by the addition of 1
.mu.g/ml of doxycycline to the culture medium for three days.
Double-resistant cells were incubated with 1 mg/ml rabbit IgG for
eighteen hours prior to staining with goat polyclonal
FITC-conjugated anti-mouse IgG (Fc.gamma.) F (ab').sub.2fragment
(Jackson ImmunoResearch Laboratories, West Grove, Pa.). Cells were
stained for 1 hour then washed twice with PBS prior to analysis by
flow cytometry. The most fluorescent cells (top 5%) were isolated
as a pool and expanded for 10 days, after which the protocol was
repeated but the top 1% most fluorescent cells were isolated as a
pool. This pool was expanded for 10 days then the top 0.1% most
fluorescent cells were isolated as single cells into 96-well
plates. Clones were analyzed by ELISA for expression of antibody
and seven clones were chosen from 53 clones analyzed. The average
specific productivity of these clones was 35 pg/cell/day and the
best clone expressed the recombinant KD5 monoclonal antibody at 54
pg/cell/day.
[0185] Although the foregoing invention has been described in some
detail by way of illustration and example, it will be readily
apparent to those of ordinary skill in the art that certain changes
and modifications may be made to the teachings of the invention
without departing from the spirit or scope of the appended
claims.
* * * * *